Photopic vision is mediated by cone cells, which function at high levels of illumination, respond to rapid changes in light (flicker) and permit color discrimination. Each cone cell expresses a protein, called a color or cone opsin, that determines its spectral sensitivity and response characteristics. Although the cone opsins are members of a larger family of visual pigments that bind 11-cis retinal to form the phototransduction molecule, and share significant amino acid sequence homology with the rhodopsins, the molecular mechanisms that produce the unique properties of cone pigments are not understood. Among the cone pigments, the short wavelength pigments (with wavelengths of peak absorbance, lambda-max equals approximately 415-440 nm) permit vision in the violet/blue region of the spectrum and are found ubiquitously in nature. In addition, responses of short wavelength cones to light show slower kinetics and increased sensitivity than the long wavelength (red) cones. In humans with severe red-green opsin mutations, the blue opsin is the sole mediator of photopic vision. Clearly, they form an important component of vision in most animals. The overall goal of this project is to understand the molecular mechanisms of 11-cis- retinal/short wavelength opsin interactions that bring about their unique absorbance properties and photobleaching/regeneration behavior; in particular, to determine how specific amino acid residues contribute to spectral tuning and phototransduction. Given the fundamental differences between rod and short wavelength cone physiology, it is not likely that direct extrapolation from rhodopsin will yield sufficient insight into the mechanistic divergence between these two different proteins. The specific aims of the proposal are to 1) biochemically characterize the bleaching/regeneration pathway and to study the physiologically active conformation of Xenopus violet cone opsin; 2) characterize the interaction of the short wavelength opsin with retinal using low temperature and time-resolved (>10 ns) UV-visible spectroscopy; 3) investigate the role specific amino acids in violet cone opsin have in retinal interactions, photobleaching and regeneration using molecular models coupled to site-directed mutagenesis.